A bismuth cluster ion-beam-based time-of-flight secondary ion mass spectrometry (TOF-SIMS) has been successfully used to image micropatterns of streptavidin and Chinese hamster ovary (CHO-k1) cells. as received and without any labeling. Three different analysis ion beams (Bi+, Bi-3(+), and Bi-3(2+)) were compared to obtain label-free TOF-SIMS chemical images of micropatterns of streptavidin, which were subsequently used for generating biotinylated cell patterns. Unlike using a Bi' ion beam, using a Bi-3(+) or Bi-3(2+), primary analysis ion beam yielded well-contrasted-TOF-SIMS images of streptavidin characteristic secondary ions. A principal component analysis of TOF-SIMS data was performed to generate a chemical image of the streptavidin itself. A chemical specific TOF-SIMS image analysis gave us a better understanding of the localization of cells at the outer boundaries of the streptavidin-patterned circles. Our work suggests that using cluster-ion analysis beams together with multivariate data analysis for TOF-SIMS chemical imaging would be an effectual method for producing label-free chemical images of micropatterns of biomolecules, including proteins and cells. (c) 2006 American Vacuum Society.